Our major goal is to study the basis of ligand recognition by GPCRs and determine how orthosteric and allosteric ligands stabilize specific receptor conformations. Together with Project 1 and 3 we will attempt to understand how ligands discriminate not only between different receptor types but to determine how they discriminate between active and inactive conformations. These endeavors will take advantage of the structural data, biophysical and pharmacological analyses to both identify novel compounds that may help to discriminate receptor isoforms. We will take into consideration divergent regions that are not involved in orthosteric, hormone-binding interactions, including the extracellular and intracellular faces ofthe receptor. We will also target regions within the TM domains but outside the orthosteric site, such as the crevice identified in the p-opiate receptor (pOR) crystal structure, between the pOR protomers of the crystallized dimer. Thus, by utilizing regions outside the orthosteric site we may engineer more selectivity into ligands that could potentially be translated to novel therapeutics. We will study how agents (small molecules, peptides and proteins) bind to- and stabilize active and inactive conformations. We will work concurrently with Projects 1 (design) and 3 (structure) to study how ligands structures generated from virtual screening around active and inactive conformations participate may stabilize, or perhaps disrupt these states. We are particularly interested in agents which display cooperative, or allosteric, effects on the hormone binding, or orthosteric site. We will take advantage of our recent crystallographic efforts to understand the basis for G protein activation has offered some insights into how G proteins allosterically alter hormone (agonist) binding.